The single layer of epithelial cells that lines the luminal surface of the intestinal mucosa are the initial site of interaction between the host and enteric microbial pathogens. The proposed studies focus on host epithelial cell responses to bacterial infection. Our overall hypothesis is that intestinal epithelial cells act as important sensors of the intestinal microbial flora, and can generate crucial host signals that orchestrate the onset of the host mucosal inflammatory response. During the prior program period, human intestinal epithelial cells were shown to upregulate the expression of an inflammatory gene program, in the early period following acute bacterial entry (i.e., within 1-3 hours). Each of the genes shown to be upregulated following bacterial invasion of intestinal epithelial cells is a targeted gene of the nuclear transcription factor NF-kappaB. By 12 hours post acute bacterial infection in vitro, intestinal epithelial cells begin to undergo apoptosis. This project proposes three Specific Aims to study early and late intestinal epithelial cell responses to bacterial infection using in vitro, and complementary in vivo, model systems. Studies in Aim 1 will define the importance of NF-kappab as a central control point, and identify additional adaptor molecules proximal to NF-kappaB that act as control points, for the activation of the intestinal epithelial cell inflammatory gene program following microbial invasion. These studies, will use in vitro systems and identify potential targets for manipulating signaling pathways that are important in the activation of the early epithelial pro-inflammatory program. The second aim is to characterize the extent to which enteroinvasive bacteria us TNFa and Fas signaling pathways to activate apoptosis in epithelial following bacterial infection. The third Aim will use three approaches to characterize signal transduction pathways in intestinal epithelial cells that are important for the activation of epithelial cell pro-inflammatory genes and epithelial cell apoptosis in vivo. Those studies will use mice transplanted with human intestinal xenografts and transgenic mice as model systems for in vivo bacterial infection. This stepwise approach to study the epithelial cell response to enteric pathogens would lead to greater understanding of the role epithelial cells can play as in integral component of a communications network that involves interactions between epithelial cells, enteric microbial pathogens, and host inflammatory and immune cells. The ability to manipulate signal transduction pathways within the intestinal epithelium could lead to new approaches for manipulating and regulating inflammatory and immune responses in the intestinal mucosa.